The present invention relates to methods of identifying compounds useful as modulators, particularly activators, of certain stress responsive kinases. More particularly, the compounds so identified are useful for treating or preventing diseases or conditions that are mediated by, for example, abnormal bone resorption or angiogenesis. These compounds are useful for treating or preventing osteoporosis, and inhibiting vascular restenosis, diabetic retinopathy, macular degeneration, angiogenesis, atherosclerosis, inflammation, and tumor growth.
It is believed that at least a dozen kinds of kinases responsive to stress exist in various mammalian cells. One of these kinases, the Mammalian Sterile 20-like Kinase has recently been described in the scientific literature. At least two isoforms of Mammalian Sterile 20-like Kinase are known, hereafter referred to as xe2x80x9cMst 1xe2x80x9d and xe2x80x9cMst 2xe2x80x9d. See, Taylor et al., xe2x80x9cNewly identified stress-responsive protein kinases, Krs-1 and Krs-2xe2x80x9d, Proc. Natl. Acad. Sci. USA, Vol. 93 (1996), pp. 10099-10104; Creasy et al., xe2x80x9cCloning and characterization of a human protein kinase with homology to Ste20,xe2x80x9d The J. of Biological Chemistry, Vol. 270, No. 37 (1995), pp. 21695-21700; Creasy et al., xe2x80x9cCloning and characterization of a member of the MST subfamily of Ste20-like kinasesxe2x80x9d, Gene, Vol. 167 (1995), pp. 303-306; Creasy et al., xe2x80x9cThe Ste 20-like protein kinase, Mst 1, dimerizes and contains an inhibitory domainxe2x80x9d, The J. of Biological Chemistry, Vol. 271, No. 35 (1996), pp. 21049-21053; and Wang and Erikson, xe2x80x9cActivation of protein serine/threonine kinases p42, p63, and p87 in Rous sarcoma virus-transformed cells: signal transduction/transformation-dependent MBP kinasesxe2x80x9d, Mol. Biol. Cell, 3, pp. 1329-1337 (1992), which are all incorporated by reference herein in their entirety. As part of the present invention, three other kinases have been identified using an in-gel kinase assay. These kinases have a molecular weight of approximately 34 kDa, 50 kDa, and 130 kDA and will be referred to herein as xe2x80x9c34 kDa Kinasexe2x80x9d, xe2x80x9c50 kDa Kinasexe2x80x9d, and xe2x80x9c130 kDa Kinasexe2x80x9d, respectively. It is believed that Mst 1, Mst 2, 34 kDa Kinase, 50 kDa Kinase and 130 kDa Kinase play a key role in regulating various mammalian cells such as osteoclasts and in regulating cellular processes such as angiogenesis.
It is believed that a wide variety of disease states and conditions can be mediated by modulating, for example by activating, Mst 1, Mst 2, 34 kDa Kinase, 50 kDa Kinase, or 130 kDa Kinase, or combinations thereof. It is also believed that so-called modulators of these kinases represent a new and useful therapeutic class of drugs. Such modulators would be useful for treating or preventing diseases which include osteoporosis, osteopenia, and Paget""s disease, and for inhibiting vascular restenosis, diabetic retinopathy, macular degeneration, angiogenesis, atherosclerosis, inflammation, and tumor growth.
In one aspect of the present invention, the activator compounds herein are useful for inhibiting bone resorption. Bone resorption is mediated by the action of cells known as osteoclasts. Osteoclasts are large multinucleated cells of up to about 400 xcexcm in diameter that resorb mineralized tissue in vertebrates. Osteoclasts are actively motile cells that migrate along the surface of bone, and can bind to bone, secrete necessary acids and proteases, thereby causing the actual resorption of mineralized tissue of the bone. More specifically, osteoclasts are believed to exist in at least two physiological states, i.e. the active/secretory state and the migratory or motile state. In the secretory state, osteoclasts are flat, attach to the bone matrix via a tight attachment, i.e. sealing, zone, become highly polarized, form a ruffled border, and secrete lysosomal enzymes and protons to resorb bone. The adhesion of osteoclasts to bone surfaces is an important initial step in bone resorption. In the migratory or motile state, the osteoclasts migrate across bone matrix and do not take part in resorption until they again attach to bone. Kinases such as Mst 1, Mst 2, 34 kDa Kinase, 50 kDa Kinase, and 130 kDa Kinase are believed to be involved in the regulation of osteoclast function. Therefore, by targeting these kinases, osteoclast mediated disease states, for example osteoporosis, can be treated.
In the present invention, it was unexpectedly discovered that certain compounds that are known to modify osteoclast function activate stress reponsive kinases. These activating compounds are the bisphosphonate alendronate, the snake venom disintegrin echistatin, anti xcex23 integrin monoclonal antibody (commercially available from Pharmingen, San Diego, Calif.), the naturally occurring polypeptide calcitonin, and the echistatin RGD (i.e. arginine, glycine, aspartate) mimetics 3(S)-(2-(2-oxo-3(S)-[5,6,7,8-tetrahydro-[1,8]-naphthyridin-2-ylmethyl)-amino]-pyrrolidin-1-yl)-acetylamino)-4-quinolin-3-yl-butyric acid trihydrochloride (hereafter xe2x80x9cRGD Ixe2x80x9d) and (2-[N-(3,4,5,6-tetrahydropyrimidin-2-yl)amino]ethyloxyphen-4-yl)carbonyl-2(S)-phenylsulfonamido-beta-alanine (hereafter xe2x80x9cRGD IIxe2x80x9d). This unexpected finding led to the further discovery of providing methods of identifying compounds which activate these kinases and the realization that other compounds so-identified would be useful for treating a variety of conditions or disease states mediated by these kinases.
It is therefore an object of the present invention to provide methods for identifying compounds which modulate a kinase responsive to stress selected from the group consisting of Mst 1, Mst 2, 34 kDa Kinase, 50 kDa Kinase, and 130 kDa Kinase and mixtures thereof.
It is another object of the present invention to provide methods of activating in mammalian cells a kinase responsive to stress selected from the group consisting of Mst 1, Mst 2, 34 kDa Kinase, 50 kDa Kinase, and 130 kDa Kinase and mixtures thereof.
It is another object of the present invention to provide methods of eliciting an activating effect in a mammal of a kinase responsive to stress selected from the group consisting of Mst 1, Mst 2, 34 kDa Kinase, 50 kDa Kinase, and 130 kDa Kinase, and mixtures thereof.
It is another object of the present invention to provide methods of treating or preventing a disease or condition in a mammal by modulating, for example activating, a kinase responsive to stress selected from the group consisting of Mst 1, Mst 2, 34 kDa Kinase, 50 kDa Kinase, and 130 kDa Kinase, and mixtures thereof.
It is another object of the present invention to provide compounds that are useful for modulating, for example activating, a kinase responsive to stress selected from the group consisting of Mst 1, Mst 2, 34 kDa Kinase, 50 kDa Kinase, and 130 kDa Kinase, and mixtures thereof.
It is another object of the present invention to provide pharmaceutical compositions comprising these compounds.
These and other objects will become readily apparent from the detailed description which follows.
The present invention relates to a method for identifying compounds which modulate a kinase responsive to stress selected from the group consisting of Mst 1, Mst 2, 34 kDa Kinase, 50 kDa Kinase, and 130 kDa Kinase, and combinations thereof, comprising:
a). contacting a putative activity-modifying compound with a cell culture selected from the group consisting of an osteoclastogenesis cell culture, purified osteoclasts, partially-purified osteoclasts, unpurified osteoclasts, purified pre-osteoclasts, partially-purified pre-osteoclasts, unpurified pre-osteoclasts, purified osteoclast-like cells, partially-purified osteoclast-like cells, unpurified osteoclasts-like cells, and mixtures thereof; and
b). determining the kinase activity of said cell culture and comparing said kinase activity with a cell culture not contacted with said putative activity-modifying compound.
In further embodiments, the present invention relates to a method of activating or otherwise modulating in a mammalian cell a kinase responsive to stress selected from the group consisting of Mst 1, Mst 2, 34 kDa Kinase, 50 kDa Kinase, and 130 kDa Kinase, and combinations thereof, comprising contacting said cell with an activating or otherwise modulating amount of a compound identified according to the methods of the present invention.
In further embodiments, the present invention relates to a method of eliciting in a mammal an activating or otherwise modulating effect of a kinase responsive to stress selected from the group consisting of Mst 1, Mst 2, 34 kDa Kinase, 50 kDa Kinase, and 130 kDa Kinase, and combinations thereof, comprising administering to said mammal an activating amount of a compound identified according to the methods of the present invention.
In further embodiments, the present invention relates to a method of treating or preventing a disease or condition in a mammal that is mediated by a kinase responsive to stress selected from the group consisting of Mst 1, Mst 2, 34 k Da Kinase, 50 kDa Kinase, and 130 kDa Kinase, and combinations thereof, comprising administering to said mammal a pharmaceutically effective amount of a compound identified according to the methods of the present invention.
In further embodiments, the present invention relates to compounds useful for activating or otherwise modulating a kinase responsive to stress selected from the group consisting of Mst 1, Mst 2, 34 kDa Kinase, 50 kDa Kinase, and 130 kDa Kinase, and combinations thereof.
In further embodiments, the present invention relates to pharmaceutical compositions comprising these compounds.